The development of vaccines directed against intracellular pathogens, for example, viruses, bacteria, protozoa, fungi, and intracellular parasites, is ongoing. The development and use of vaccines has proved invaluable in preventing the spread of disease in man. For example, in 1967, smallpox was endemic in 33 countries with 10 to 15 million cases being reported annually. At that time, the World Health Organization introduced a program to eradicate smallpox. Approximately one decade later, smallpox was successfully eradicated from the human population.
Theoretically, an ideal vaccine has a long shelf life, is capable of inducing with a single dose long lasting immunity against a preselected pathogen and all of its phenotypic variants, is incapable of causing the disease to which the vaccine is directed against, is effective therapeutically and prophylactically, is prepared easily and economically using standard methodologies, and can be administered easily in the field.
Presently four major classes of vaccine have been developed against mammalian diseases. These include: live-attenuated vaccines; non living whole vaccines; vector vaccines; and subunit vaccines. Several reviews discuss the preparation and utility of these classes of vaccines. See for example, Subbarao et al. (1992) in Genetically Engineered Vaccines, edited by Ciardi et al., Plenum Press, New York; and Melnick (1985) in High Technology Route to Virus Vaccines, edited by Dreesman et al., published by the American Society for Microbiology, the disclosures of which are incorporated herein by reference. A summary of the advantages and disadvantages of each of the four classes of vaccines is set forth below.
Live attenuated vaccines comprise live but attenuated pathogens, i.e., non-virulent pathogens, that have been "crippled" by means of genetic mutations. The mutations prevent the pathogens from causing disease in the recipient or vaccinee. The primary advantage of this type of vaccine is that the attenuated organism stimulates the immune system of the recipient in the same manner as the wild type pathogen by mimicking the natural infection. Furthermore, the attenuated pathogens replicate in the vaccinee thereby presenting a continuous supply of antigenic determinants to the recipient's immune system. As a result, live vaccines can induce strong, long lasting immune responses against the wild type pathogen. In addition, live vaccines can stimulate the production of antibodies which neutralize the pathogen. Also they can induce resistance to the pathogen at its natural portal of entry into the host. To date, live attenuated vaccines have been developed against: smallpox; yellow fever; measles; mumps; rubella; poliomyelitis; adenovirus; and tuberculosis.
Live attenuated vaccines, however, have several inherent problems. First, there is always a risk that the attenuated pathogen may revert back to a virulent phenotype. In the event of phenotypic reversion, the vaccine may actually induce the disease it was designed to provide immunity against. Second, it is expensive and can be impractical to develop live vaccines directed against pathogens that continuously change their antigenic determinants. For example, researchers have been unable to develop a practical live vaccine against the influenza virus because the virus continually changes the antigenic determinants of its coat proteins. Third, live attenuated vaccines may not be developed against infections caused by retroviruses and transforming viruses. The nucleic acids from these viruses may integrate into the recipients genome with the potential risk of inducing cancer in the recipient. Fourth, during the manufacture of live attenuated vaccines adventitious agents present in the cells in which the vaccine is manufactured may be copurified along with the attenuated pathogen. Alien viruses that have been detected in vaccine preparations to date include the avian leukosis virus, the simian papovavirus SV40, and the simian cytomegalovirus. Fifth, live vaccine preparations can be unstable therefore limiting their storage and use in the field. Presently, attempts are being made to develop stabilizing agents which enhance the longevity of the active vaccines.
Non living whole vaccines comprise non viable whole organisms. The pathogens are routinely inactivated either by chemical treatment, i.e., formalin inactivation, or by treatment with lethal doses of radiation. Non living whole vaccines have been developed against: pertussis; typhus; typhoid fever; paratyphoid fever; and particular strains of influenza.
In principle, non living vaccines usually are safe to administer because it is unlikely that the organisms will cause disease in the host. Furthermore, since the organism is dead the vaccines tend to be stable and have long shelf lives. There are, however, several disadvantages associated with non living whole vaccines. First, considerable care is required in their manufacture to ensure that no live pathogens remain in the vaccine. Second, vaccines of this type generally are ineffective at stimulating cellular responses and tend to be ineffective against intracellular pathogens. Third, the immunity elicited by non viable vaccines is usually short-lived and must be boosted at a later date. This process repeatedly entails reaching the persons in need of vaccination and also raises the concern about hypersensitizing the vaccinee against the wild type pathogen.
Vector vaccines, also known as live recombinant vehicle vaccines, may be prepared by incorporating a gene encoding a specific antigenic determinant of interest into a living but harmless virus or bacterium. The harmless vector organism is in turn to be injected into the intended recipient. In theory, the recombinant vector organism replicates in the host producing and presenting the antigenic determinant to the host's immune system. It is contemplated that this type of vaccine will be more effective than the non-replicative type of vaccine. For such a vaccine to be successful, the vector must be viable, and be either naturally non-virulent or have an attenuated phenotype.
Currently preferred vectors include specific strains of: vaccinia (cowpox) virus, adenovirus, adeno-associated virus, salmonella and mycobacteria. Live strains of vaccinia virus and mycobacteria have been administered safely to humans in the form of smallpox and tuberculosis (BCG) vaccines, respectively. They have been shown to express foreign proteins and exhibit little or no conversion into virulent phenotypes. Several types of vector vaccines using the BCG vector currently are being developed against the human immunodeficiency virus (HI). For example, the HIV antigenic proteins: gag; env; HIV protease; reverse transcriptase; gp120 and gp41 have been introduced, one at a time, into the BCG vector and shown to induce T cell mediated immune responses against the HIV proteins in animal models (Aldovini et al. (1991) Nature 351:479-482; Stover et al. (1991) Nature 351:456-460; Colston (1991) Nature 351:442-443).
Vector vaccines are capable of carrying a plurality of foreign genes thereby permitting simultaneous vaccination against a variety of preselected antigenic determinants. For example, researchers have engineered several HIV genes into the vaccinia virus genome thereby creating multivalent vaccines which therefore are, in theory, capable of simultaneously stimulating a response against several HIV proteins.
There are several disadvantages associated with vector vaccines. First, it is necessary to identify suitable strains of viable but non-pathogenic organisms that may act as carriers for the genes of interest. Second, vector vaccines can be prepared only when a potentially protective antigenic determinants has been identified and characterized. Accordingly, vector vaccines cannot be prepared against pathogens whose antigenic determinant has not yet been identified or are so variable that the prospect of identifying the antigenic determinant for each variant is impractical. Third, the genes encoding the preselected antigenic determinant must be stably transfected and expressed in the preferred carrier organism. Consequently, the methodologies required for developing this type of vaccine are both labor intensive and time consuming. Fourth, it has not yet been established that recombinant vector vaccines effectively immunize a recipient against a preselected pathogen.
Subunit vaccines usually comprise a subcellular component purified from the pathogen of interest. Subunit vaccines usually are safe to administer because it is unlikely that the subcellular components will cause disease in the recipient. The purified subcellular component may be either a defined subcellular fraction, purified protein, nucleic acid or polysaccharide having an antigenic determinant-capable of stimulating an immune response against the pathogen. The antigenic components can be purified from a preparation of disrupted pathogen. Alternatively, the antigenic proteins, nucleic acids or polysaccharides may be synthesized using procedures well known in the art. Diseases that have been treated with subunit type vaccines include: cholera; diphtheria; hepatitis type B; poliomyelitis; tetanus; and specific strains of influenza.
There are, however, several disadvantages associated with subunit vaccines. First, it is important to identify and characterize the protective antigenic determinant. This can be a labor intensive and time consuming process. As a result it may be impractical to develop subunit vaccines against pathogens with highly variable antigenic determinants. Second, subunit vaccines generally are ineffective at stimulating cytotoxic T cell responses and so they may be ineffective at stimulating an immune response against intracellular pathogens. Third, the immunity elicited by subunit vaccines is usually short-lived, and like the non living whole vaccines must be boosted at a later date therefore raising the concern about hypersensitizing the vaccinee against the wild type pathogen.
Heretofore, many of the inactivated whole and subunit vaccines have not been sufficiently immunogenic by themselves to induce strong, protective responses. As a result, immunostimulants including, for example, aluminum hydroxide; intact mycobacteria; and/or mycobacterial components have been co-administered with these vaccines to enhance the immune response stimulated by the vaccine. Recently, experiments have shown that mycobacterial heat shock proteins may act as carriers for peptide vaccines thereby enhancing the immunogenicity of the peptides in vivo (Lussow et al. (1991) Eur. J. Immnunol. 21:2297-2302). Further studies have shown that administering a composition to mice comprising an antigenic peptide chemically crosslinked to a purified mycobacterial stress protein stimulates a humoral (antibody mediated) rather than a temporal (cell mediated) response against the antigenic peptide (Barrios et al. (1992) Eur. J. Immunol. 22.1365-1372).
However, because it is generally believed that cellular responses are required for immunizing against intracellular pathogens (see for example, "Advanced Immunology," Male et al. (1991) Gower Medical Publishing; Raychaudhuri et al. (1993) Immunology Today 14:344-348) it is contemplated that conventional subunit and inactivated whole organism vaccines may be ineffective at stimulating immune responses, specifically cytotoxic T cell responses, against intracellular pathogens.
It is an object of the instant invention to provide a safe subunit vaccine comprising a stress protein-peptide complex for administration to a mammal that is capable of inducing, by means of a cytotoxic T cell response, resistance to infection by a preselected intracellular pathogen. The vaccines prepared in accordance with the invention may be used to elicit an immune response against an intracellular pathogens whose antigenic determinants have been identified, have not yet been identified, or where it is impractical to isolate and characterize each of the antigenic determinants. The vaccines prepared in accordance with the invention may be prophylactically and therapeutically effective against preselected pathogens.
Another object of the invention is to provide a method for inducing in a mammal resistance to infection by an intracellular pathogen by administering to the mammal a stress protein-peptide subunit vaccine. Another object is to provide a method for rapidly and cost effectively producing commercially feasible quantities of the stress protein-peptide vaccines from a cell or cell line infected with the intracellular pathogen or alternatively from a cell or cell line transfected with, and expressing a gene encoding a specific antigenic determinant. Still another object is to provide a method for preparing an immunogenic stress protein-peptide subunit vaccine by reconstituting in vitro immunologically unreactive stress proteins and peptides thereby to produce immunoreactive complexes capable of stimulating an immune response against a preselected intracellular pathogen.
These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.